1. Field of the Invention
The present invention generally relates production of recombinant clusterin and the use thereof for prevention and treatment of pathological conditions.
2. Description of Related Art
Hypercholesterolemia represents the most defined risk factor for atherosclerosis, an arterial disease that causes myocardial and cerebral infarctions. Low density lipoprotein (LDL) carries cholesterol from the liver to peripheral tissues, and when elevated in blood, LDL deposits the lipid in the arterial wall, which in turn develops atherosclerotic plaques and increases the risk for thrombogenic events in the arteries. In contrast, high density lipoprotein (HDL) functions as a reverse cholesterol-transporter that removes the lipid from the arterial wall to the liver where cholesterol is metabolized. In essence, LDL is pro-atherogenic while HDL anti-atherogenic (Nicholls et al., 2005; Ansell et al., 2004; von Eckardstein et al., 2005) LDL mainly contains ApoB 100 and HDL apoE and apoA-I. In spite of the success of lowering LDL-cholesterol therapy with statins, raising HDL levels with torcetrapib (an inhibitor of cholesterol ester transfer protein (CETP) has shown little benefit to patients with atherosclerosis (Kastelein et al., 2007; Nissen et al., 2007). The failure of torcetrapib therapy underscores the incompleteness of our fundamental understanding of HDL function. HDL particles are heterogeneous in shape, density, size, composition and have multiple functional properties such as reverse cholesterol transport, as well as anti-oxidant, anti-inflammatory, and anti-thrombotic activities. Indeed, dysfunctional, proinflammatory HDL has been found in several pathological conditions, including atherosclerosis (Smith et al., 2010), diabetes (Hoofnagle et al. 2010) and autoimmune disorders (McMahon et al., 2009; Volkmann et al., 2010). Thus, the development of an anti-atherogenic, anti-apoptotic, and anti-inflammatory agent that enables HDL beneficial action is of clinical significance.
Clusterin is a sulfated, heterodimeric glycoprotein containing two 40 kDa chains joined by a unique five disulfide bond motif 1). It contains several domains, such as amphipathic helix, heparin-binding domain, and lipid-binding domain. This protein was initially identified from ram rete testes fluid and named for its ability to elicit clustering of Sertoli cells supporting sperm maturation and development (NCBI/GenBank Accession No. NM_203339, NM_001831) Thereafter, species homologues have been isolated and cloned by a number of groups working in widely divergent areas, generating various synonyms of clusterin, including testosterone repressed prostate message-2 (TRPM-2), sulfated glycoprotein-2 (SGP-2), apolipoprotein-J (Apo-J), SP-40, 40, complement cytolysis inhibitor (CLI), and dimeric acidic glycoprotein (DAG), gp 80, NA1/NA2, glycoprotein III, etc.
Encoded on a 2-kb mRNA, clusterin is transcribed from a single copy gene located on mouse chromosome 14 and human 8p21 (Fink et al., 1993), and translated as a 51 kD or so protein comprising 427 amino acid sequence (Jordan-Starck et al., 1994). In the blood stream, clusterin circulates mainly with HDL as one of apolipoproteins (Choi-Miura et al., 1992; Stuart et al., 1992) but a small portion of clusterin may exist in LDL (Karlsson et al., 2005). Clusterin expression is induced by stress responses (Wilson et al., 2000). Clusterin binds megalin/LRP-2 receptor, members of LDL receptor family. Increased expression of clusterin occurs in both human (Mackness et al., 1997; Ishikawa et al., 2001; Ishikawa et al., 1998) and experimental animal (Jordan-Starck et al., 1994; Navab et al., 1997) atherosclerotic lesions. Reported functions of clusterin include apoptosis inhibition (Kowolik et al. 2006), complement factor inactivation (Correa-Rotter et al., 1992), lipid recycling and transport (Gelissen et al., 1998), membrane protection, and maintenance of cell-cell or cell-substratum contacts. It can effectively bind to lipids and promote efflux of cholesterol and oxysterols from lipid-laden foam cells, a hall-marker of atherosclerosis (Gelissen et al., 1998). Clusterin has a high-affinity to a wide array of biological ligands. The presence of both hydrophilic and hydrophobic domains enables clusterin to act as a chaperone or a “biological detergent”.
Clusterin plays a role in regulation of metabolism and function of various tissues and organs, particularly in the cardiovascular system. HDL with decreased levels of clusterin has been found in association with a high incidence of myocardiac infarction in patients with insulin-resistant metabolic syndromes (Hoofnagle et al., 2010). Administration of an oral clusterin peptide was reported to reduce atherosclerosis in ApoE-null mice (Navab et al., 2005), and intravenous injection of clusterin diminishes rat myocardiac infarction (Van Dijk et al., 2010). Transduction of clusterin can restore the mitochondrial membrane potential and prevent the release of cytochrome-c from mitochondria into cytoplasma in cardiac myoblasts damaged by ethanol (Li et al., 2007). Furthermore, increased clusterin expression in myoblasts enhances the cell capacity of migration and homing through induction of CXCR4, a chemokin-receptor for stromal cell-derived factor (SDF) (Li et al., 2010).
Human clusterin gene located in chromosome 8 (location 8p21-p18) with 17876 by long contains 10 exons in total. Exon one and exon two are alternative yielding two different transcript isoforms. Other exons (Ansell et al., 2004; von Eckardstein et al., 2005; Kastelein et al., 2007; Nissen et al., 2007; Smith et al., 2010; Hoofnagle et al. 2010; McMahon et al., 2009; Volkmann et al., 2010) are shared with both isoforms. clusterin transcripts contain 3 different translation start sites (ATG), all in-frame. The best characterized protein isoform is produced from transcript isoform 2, where translation starts at the second ATG present in exon 2, right before ER-targeting signal. clusterin protein precursor (NP-976084) consists of 449 amino acids. There is evidence suggesting that two nuclear protein isoforms can be produced from this transcript isoform, one in which translation starts at ATG in exon 3 (417 aa), and another with translation starting from ATG in exon 1 (459 aa). Secreted clusterin is produced from the transcript isoform 2. The initial protein precursor, presecretory psCLU (˜60 kDa), becomes heavily glycosylated and cleaved in the ER, and the resulting alpha and beta peptide chains are held together by 5 disulfide bonds in the mature secreted heterodimer protein form, sCLU (˜75-80 kDa).
Under stimulation by ionic radiation and oxidative stress, the nuclear clusterin is first translated as a non-glycosylated protein precursor, pnCLU (˜49 kDa), that is then translocated into nucleus. There is evidence of two distinct sized nuclear clusterin proteins (˜50 kDa and ˜60 kDa) (Pajak et al., 2007), that could result from translation started either at ATG present in exon 3 or in exon 1, respectively. Secreted clusterin is cytoprotective but nuclear clusterin cytotoxic. The controversy of clusterin functions mainly results from the not well-established role of the two different protein isoforms with distinct subcellular localization and somewhat opposing functionalities. Some known functions include involvement in apoptosis through complexing with Ku70 autoantigen (nCLU, proapoptotic) or interfering with Bax-activation (sCLU, antiapoptotic) (Araki et al., 2005; Klokov et al., 2004; Leskov et al., 2003; Yang et al., 2000). Clusterin has also been linked to spermatogenesis, epithelial cell differentiation, TGF-beta signaling through Smad2/Smad3 (Shin et al., 2008; Ahn et al., 2008; Lee et al., 1992), complement activation (Dietzsch et al., 1992; O'Bryan et al., 1990). Secreted native clusterin contains the sequence domains of nuclear clusterin critical for nuclear translocation and binding to nuclear death signaling proteins such as Ku70.
Despite the various roles in cellular regulation ascribed to clusterin, there remains a need for the development of recombinant clusterin and clusterin analogs as potential therapeutics. Embodiments of this invention disclose technology of producing recombinant clusterin with a high homology to the secreted form of native clusterin with a protective function, and compositions of recombinant clusterin that can be used for prevention and treatment of diseases in a mammal.